Table 2 Steady state kinetic parameters and relative amounts of sesquiterpenes produced by PR-AS, PR-AS-L108A, PR-AS-L108S, PR-AS-L108V and
PR-AS-L108F
Enzyme
KM (mM)
kcat (¥10-3 s-1)
84
0.9 0.2
9.5 0.2
5.7 0.3
3.6 0.1
k
cat/KM (¥10-3 s-1 mM-1)
4 (%)
2 (%)
5 (%)
6 (%)
7 (%)
8 (%)
PR-AS
0.53 0.2
0.70 0.2
0.99 0.1
2.03 0.3
1.76 0.1
5
158.49 0.71
1.29 0.47
9.60 0.10
2.81 0.07
2.05 0.03
91.5
6.8
9.4
88.3
74.1
7.5
13.9
14.6
8
1
0
0
0
a
PR-AS-L108A
PR-AS-L108S
PR-AS-L108V
PR-AS-L108F
26.9
21.5
0
48.2
49.1
0
4.1
5.3
2.3
0
a
1
12
13.8
0
0
a Trace amounts.
Clearly, the flexible prenyl chain of FDP binds to the altered
active sites of PR-AS-L108A and PR-AS-L108S in a more
extended conformation, in which C1 and C10 are no longer held
sufficiently close for macrocyclization. Interestingly, the product
distributions observed with PR-AS-L108A and PR-AS-L108S
were similar to that obtained from the unselective Pd(0)-catalyzed
elimination of farnesyl acetate under mild conditions (Scheme 2
and ESI†).40
Notes and references
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Since the reduction in the van der Waals volume induced by
the L108A and L108S replacements is expected to compromise
a possible interaction between Phe 112 and the aliphatic Leu
108 that would lead to an increase of cyclic products (Table 2),
it is tempting to suggest that Leu 108 interacts with Phe 112
to establish the active site-contour that enforces the productive
conformation of FDP essential for aristolochene biosynthesis. In
agreement with this proposal, the replacement of Leu 108 with
valine, which has almost twice the van der Waals volume of
alanine, resulted in a product distribution that closely matched
that of PR-AS (in addition to a small amount (~2%) of the
acyclic 8). Finally, replacement of Leu 108 with the significantly
larger phenylalanine led to the accumulation of cyclic products
(4, 2 and 5) arising from the native C1–C10 ring closure. Taken
together, these observations suggest that the productive, quasi-
cyclic precatalytic conformation of FDP (1) is maintained so long
as sufficient steric bulk is available. These observations underline
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residue Leu 108 for aristolochene synthase.
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in PR-AS catalysis of an aliphatic residue (Leu 108), which
is deeply buried in the lower, hydrophobic active site cleft.
Several aromatic (Tyr 92, Phe 112 and possibly Phe178) and
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BB/G003572/1 and BB/H01683X/1 and by the Engineering and
Physical Sciences Research Council through grant EP/D06958/1.
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6922 | Org. Biomol. Chem., 2011, 9, 6920–6923
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